Program Director/Principal Investigator (Last, First, Middle): Baker, Timothy 1R01 AI079095-01 Project Summary Modern electron cryo-microscopy and computer-based, 3D image reconstruction techniques are revolutionizing the way structures of large and complex biomacromolecular machines are studied. These methods provide keys to understanding at the molecular level how these machines function. The scientific goal of our studies is to determine virus structures reliably at the highest possible resolutions and in the shortest amount of time, as this will enhance our ability to understand how viruses cause a variety of diseases. Such structural work may yield important clues about how best to develop anti-virals for preventing viral infections. Microscopy and image reconstruction each pose their own set of significant challenges, and continued development of our robust, efficient software clearly represents a key to the success of our research efforts as well as that of others. This proposal aims to substantially enhance the computational capabilities that comprise the heart of the 3D structure determination part of our cryo-reconstruction work. The ultimate goal of the proposed work is to perform 3D image reconstructions of icosahedral viruses directly at the microscope in near real-time. Over the next two years, we realistically expect to reach low resolutions (25-30A) in less than five minutes after a micrograph has been collected. This will be accomplished through improvements to the speed and parallel scalability of the numerically intensive applications, enhancements to our automated image reconstruction system, and modifications to existing image boxing software so that individual virus particles can be identified without manual intervention. We also plan to improve the reliability of the random model method for constructing a starting model de novo from unaligned images when a suitable starting map is not available. Even in instances when the reconstructions are not done at the microscope, this work will greatly streamline and accelerate structure determination projects. In conjunction with the efforts described above, we will implement algorithmic changes that make it possible to reach higher resolutions. These new capabilities are expected to have a profound impact on many problems of biological interest since they allow one to rapidly screen samples for desired properties. Current challenges in our lab include determining the locations of Nanogold-labeled cysteine residues in P22 mutants, the presence or absence of "flaps" in the vicinity of 5-fold symmetry axes in baboon reovirus, binding stoichiometry of antibodies and other ligands to the surface of human parvoviruses, and gross morphological changes due to smalw I 9 residue) insertions in the E2 glycoprotein of alphaviruses. The proposed work is heavily leveraged by close ties to colleagues in cryo-microscopy, image processing, and data visualization and interpretation, the recent acquisition of two new microscopes with NIH support, and interactions with the San Diego Supercomputer Center. All of our software will be made readily accessible to the electron microscopy community. PHs 39812590 (Rev. 11/07) Page 2 Continuation Format Page